Careful orchestration of mRNA synthesis is critical for programmed cell death, normal cell growth and development, and maintenance of health and well-being in humans. The broad, long term objectives of this project are to understand the mechanisms that underlie the intricate regulation of eukaryotic gene expression. Understanding how gene expression occurs in a timely and coordinate manner requires detailed knowledge of the components of the transcriptional machinery. The heart of this machinery comprises the RNA polymerase II holoenzyme, a multiprotein complex that contains RNA polymerase II. This proposal focuses on the study of five of the holoenzyme components, the RNA polymerase II subunits designated RPB4, RPB5, RPB6, RPB7 and RPB9, to help identify their roles in transcription. Specific Aim l involves making conditional mutants in each of these subunits and testing them for transcriptional defects using several in vivo and in vitro assays.- In Specific Aim II, mutants affecting initiation, elongation or termination will be subjected to suppressor analysis in order to uncover novel interacting proteins and identify known transcription factors that directly contact RNA polymerase. To complement this genetic approach, two- hybrid screens with the wild-type subunits will be used to identify additional interacting proteins that may not be revealed by genetic suppression experiments. In Specific Aim III the biological significance of RPB6 phosphorylation will be investigated. Potential phosphorylation sites at ten conserved residues will be altered using site-directed mutagenesis. The mutants will be subjected to in vitro transcription experiments and other in vivo studies. Phosphorylation site-specific antibodies will also be enlisted to help pinpoint the location of the target site. Finally, kinases that are involved in RPB1-carboxyl terminal domain phosphorylation and other candidate kinases will be tested for potential roles in RPB6 phosphorylation and function.